Gastrodin ameliorates cognitive dysfunction in diabetes by inhibiting PAK2 phosphorylation.

Diabetes is associated with higher prevalence of cognitive dysfunction, while the underlying mechanism is still elusive. In this study, we aim to explore the potential mechanism of diabetes-induced cognitive dysfunction and assess the therapeutic effects of Gastrodin on cognitive dysfunction. Diabetes was induced by a single injection of streptozotocin. The Morris Water Maze Test was employed to assess the functions of spatial learning and memory. Transcriptome was used to identify the potential factors involved. Western blot and immunofluorescence were applied to detect the protein expression. Our results have shown that spatial learning was impaired in diabetic rats, coupled with damaged hippocampal pyramidal neurons. Gastrodin intervention ameliorated the spatial learning impairments and neuronal damages. Transcriptomics analysis identified differential expression genes critical for diabetes-induced hippocampal damage and Gastrodin treatment, which were further confirmed by qPCR and western blot. Moreover, p21 activated kinase 2 (PAK2) was found to be important for diabetes-induced hippocampal injury and its inhibitor could promote the survival of primary hippocampal neurons. It suggested that PAK2 pathway may be involved in cognitive dysfunction in diabetes and could be a therapeutic target for Gastrodin intervention.

Induction of lung epithelial cell transformation and fibroblast activation by Yunnan tin mine dust and their interaction.

Tumor-stroma interactions play a significant role in tumor development and progression. Our study employed an in vitro co-culture model of epithelial cells and fibroblasts to investigate the mechanism of and interaction between lung epithelial cell transformation and fibroblast activation induced by Yunnan tin mine dust. Epithelial cell transformation was evaluated using concanavalin A agglutination and anchorage-independent growth assays, and fibroblast activation was assessed via immunohistochemistry. The TGF-ß1/Smad pathway was monitored by Western blot analysis and ELISA. We found concanavalin A agglutination and anchorage-independent growth assays of dust-exposed epithelial cells were positive, dust-exposed fibroblasts expressed α-SMA, and during the mine dust-induced tumorigenesis, TGF-ß1/Smad signaling pathway changed. In conclusion, Yunnan tin mine dust is able to induce the malignant transformation of bronchial epithelial cells and fibroblast activation. Epithelial cells are the main target of mine dust. Bronchial epithelial cell transformation and fibroblast activation are correlated and synergistic. Their interdependence is related to the TGF-ß1/Smad signaling pathway.

[Expression of fragile histidine triad (FHIT) protein and Ki-67 in transformed epithelial cells induced by Yunnan tin mine dust].

OBJECTIVE: To study the expression and significance of fragile histidine triad (FHIT) and Ki-67 in transformed epithelial cells induced by Yunnan tin mine dust. METHODS: Every second generation of immortalized human bronchial epithelial cells (BEAS-2B) and human embryo lung fibroblasts (WI-38) were exposed to 100 µg/ml Yunnan tin mine dust for 72 h, until the ninth generation. The cells were subsequently co-cultured from the 11th generation. Experimental setup: B group, B (W) group, B (W 100) group, B100 group, B100 (W) group, B100 (W100) group. The expressions of FHIT and Ki-67 in epithelial cells were determined by the method of immunocytochemistry at the 16th, 26th and 36th generation. The percentage of Ki-67 positive cells was calculated as proliferation index. RESULTS: The expression of FHIT was observed in BEAS-2B cells. The expression levels of FHIT among B group, B (W) group and B (W 100) group had not instinctive difference. At the 16th generation, the expression of FHIT in the B100 group was decreased compared with that in the B group and the expression of FHIT between B100 (W) group and B100 (W100) group was lower than that in the B100 group. At the 26th generation, the expression of FHIT was decreased compared with that at the 16th generation in the B100, B100 (W) and B100 (W100) groups. However, At the 36th generation, positive expression were observed again in the B100, B100 (W) and B100 (W100) groups and the expression levels were in incremental order. At the 16th, 26th and 36th generation, the proliferation indexes of B group, B (W) group and B (W 100) group were all < 3%. The proliferation indexes of B100, B100 (W) and B100 (W100) were increased step by step with the generation elongation. CONCLUSIONS: FHIT could be a target at which Yunnan tin mine dust induces transformation of BEAS-2B cells. The proliferation activation of BEAS-2B cells can be improved by Yunnan tin mine dust.

[Interaction between malignant transformation of human pulmonary epithelial cells and activation of fibroblasts induced by Yunnan tin mine dust].

OBJECTIVE: To study the interaction between transformation of human pulmonary epithelial cells and activation of fibroblasts induced by Yunnan tin mine dust. METHODS: (1) The immortalized human bronchial epithelial cell line BEAS-2B and human embryo lung fibroblast cell line WI-38 were grown in MEM medium containing 5% and 10% FBS, respectively, at 37 degrees C and 5% CO2 with saturated humidity. The cells were subcultured every 6 days. BEAS-2B cells and WI-38 cells were induced with Yunnan tin mine dust on every other generation at the concentration of 100 microg/ml. From the 11th generation, the cells were co-cultured. Epithelial cell transformation was tested using concanavalin A (ConA) agglutination and anchorage-independent growth assays. The cell cycles were analyzed through flow cytometry. The expressions of alpha-SMA in fibroblasts were determined with immunocytochemistry. RESULTS: (1) Cell morphology of mine dust-exposed epithelial cells began to transform at the 28th generation. Similar transformations were observed with mine dust-induced epithelial cells co-cultured with fibroblasts from the 20th generation and mine dust-induce epithelial cells co-cultured with mine dust-induced fibroblasts from the 16th generation. ConA agglutination assay and anchorage-independent growth assays were negative in normal BEAS-2B cells. At the 26 th generation, the agglutination test result of the mine dust-exposed epithelial cells was positive. Co-cultured with fibroblasts and mine dust-exposed fibroblasts, the agglutination time of the mine dust-exposed epithelial cells became short. Epithelial cell anchorage-independent growth assay was positive for mine dust-exposed epithelial cells co-cultured with fibroblasts at the 36th generations and for mine dust-exposed epithelial cells co-cultured with mine dust-exposed fibroblasts at the 26th generations. The clone formation rate of the 26th generation was 6.00 per thousand +/- 1.00 per thousand and 15.33 per thousand +/- 2.52 per thousand respectively, with the significant differences (P < 0.05). With generation adding, the portion of S phase increased for mine dust-exposed epithelial cells. (2) At the 26th generations, fibroblasts expressed alpha-SMA. Co-cultured with epithelial cell, the alpha-SMA expression of fibroblasts increased. Especially, positive cell numbers and intensity of staining dramatically increased with generation adding. CONCLUSIONS: (1) The tin mine dust can induce malignant transformation of human pulmonary epithelial cells BEAS-2B and activation of fibroblasts WI-38. (2) The epithelial cells are major target in carcinogenesis induced by Yunnan tin mine dust. (3) Transformation of epithelia and activation of fibroblasts co-evolve in the developing process of induced lung cancer by Yunnan tin mine dust.

[Expression of p14(ARF) and E2F-1 in lung cancer in Gejiu and Xuanwei regions of Yunnan Province].

OBJECTIVE: To investigate the correlations between p14(ARF) and E2F-1, and the role of their alterations in the tumorigenesis of the lung cancer in Gejiu and Xuanwei regions in Yunnan Province for providing the important experiment basis in revealing the molecular mechanism and looking for new markers for early diagnosis of lung cancer. METHODS: The expression of p14(ARF) and E2F-1 was detected at theirs protein level by Immunohistochemistry S-P method in 30 specimens of lung cancer of Gejiu tin miners, 30 specimens of lung cancer of Xuanwei peasants and 20 specimens of normal lung tissue. E2F-1 mRNA was detected by ISH in 25 specimens of lung cancer of Gejiu tin miners, 25 specimens of lung cancer of Xuanwei peasants and 10 specimens of normal lung tissue. The positive signals were quantitatively analysed by HPIAS-100. RESULTS: The positive unit (PU) of p14(ARF) and E2F-1 was 16.44 +/- 4.85 and 47.39 +/- 5.43 in Gejiu group, and 16.79 +/- 3.55 and 48.15 +/- 9.11 in Xuanwei group. Expression of p14(ARF) and E2F-1 protein in lung cancer of Gejiu and Xuanwei were statistically different compared with that in the normal lung (P < 0.01) respectively; The PU of E2F-1 mRNA was 48.58 +/- 7.75 in Gejiu group, and 49.41 +/- 8.53 in Xuanwei group, which was higher than that in normal tissue group. The differences were significant (P < 0.01). There was positive correlation between the expression of E2F-1 protein and E2F-1 mRNA in Gejiu group, Xuanwei group and normal group (P < 0.01, r = 0.833). The expression of p14(ARF) protein was significantly negatively correlated with the expression of E2F-1 protein (P < 0.01, r = -0.830). CONCLUSION: There is the over-expression of E2F-1 gene and the deletion of p14(ARF) gene in the tumorigenesis of the lung cancer in Gejiu and Xuanwei regions in Yunnan Province. Over-expression of E2F-1 protein in lung cancer may be caused by enhanced transcription.

[Expression of hTERT and Mad1 in lung cancer in Gejiu and Xuanwei of Yunnan Province].

OBJECTIVE: To study the expression of hTERT mRNA and Mad1 protein in lung cancer of Gejiu and Xuanwei and normal lung tissue and to investigate their correlations with lung cancer. METHODS: Mad1 protein was detected by immunohistochemistry S-P method, and hTERT message RNA (mRNA) was detected by in situ hybridization (ISH) in 40 specimens of lung cancer of Gejiu Tin miners and 20 specimens of lung cancer of Xuanwei peasants and 20 specimens of normal lung tissue. The positive signals were quantitatively analyzed by HPIAS-100. RESULTS: The positive unit (PU) of Mad1 protein was 16.77 +/- 6.01 in Gejiu Tin Miners lung cancer group, and 19.36 +/- 4.54 in Xuanwei peasant lung cancer group, compared with the normal lung tissue (46.05 +/- 7.26). The difference was highly significant (P < 0.01); The PU of hTERT mRNA was 72.10 +/- 13.07 in Gejiu Tin miners lung cancer group, and 74.20 +/- 15.17 in Xuanwei peasant lung cancer group, which was higher than that in normal tissue group (10.70 +/- 2.21). The difference was significant (P < 0.01). The expression of Mad1 protein was negatively correlated with the expression hTERT mRNA (P < 0.05, r = 0.9881, r = -0.999). CONCLUSION: Reduced hTERT mRNA expression may play an important role in the occurrence of lung cancer. The expression of hTERT mRNA and deletion of Mad1 protein are closely related to pathogenesis of lung cancer.

[Pathological study of lung cancer induced by Yunnan tin mine dusts in F344 rats].

OBJECTIVE: To set up animal models of the lung cancer induced by Yunnan tin mineral dusts (no radon) in F344 rats and to explore the process of carcinogenesis and pathologic alterations in various stages of malignant transformation in the animal models. METHODS: One hundred and ninety F344 rats were randomly divided into Yunnan tin mineral dust group (100 rats), furfural physiological saline group (30 rats), physiological saline group (30 rats) and normal control group (30 rats). The intratracheal instillation with mass fraction of 6% suspension liquid mixture Yunnan tin mineral dusts, volume fraction of 2% furfural physiological saline and physiological saline 0.2 ml was performed in the rates once per week respectively except normal control group. Then the rats were sacrificed in batch periodically after one week. The last rat was exposed to the tin mine dusts for 100 weeks. The morphological process and tumor formation were dynamically observed under LM and TEM. Immunohistochemistry detection of cytokeratin of High MW and low MW was used for tumor classification. Pollak stein was used to evaluate the development of fibrosis of lung in the rats. RESULTS: Bronchoalveolar inflammation occurred in the early stage after the intratracheal instillation of Yunnan tin mineral dust was performed in F344 rates. Along with reduction of inflammation, collagen fibrils increased at alveolar interstices. Simple hyperplasia, papillary hyperplasia and metaplasia of the epithelial cells in alveolar and bronchi were observed, followed by atypical adenomatous hyperplasia and squamous dysplasia. Lung cancer was induced in the end. Among the 14 cases of lung cancer, 9 cases were adenocarcinoma, 2 squamous cell carcinoma and 3 mixed carcinoma. No lung cancer occurred in other three control groups. There was a significant difference in the malignancy rate between the experimental group and the three control groups (P < 0.01). The squamous metaplasia and squamous carcinoma were found in alveoli that expressed cytokeratin of High MW. Lung fibrosis was found in 31 cases of in the tin mineral dust group. The greater the mineral dust deposit was, the more serious the alveolar fibrosis was. CONCLUSION: Yunnan tin mineral dusts without radon induce lung cancer in rates. The adenocarcinoma and squamous carcinomas induced in F344 rat lung can occur in the alveoli. The further study on whether type II alveolar epithelial cells are the origin cells of adenocarcinoma and some peripheral squamous lung carcinomas is worthwhile.

[Loss of exons of FHIT gene and FHIT protein expression in Yunnan tin miners with lung cancer].

BACKGROUND & OBJECTIVE: Yunnan tin miners have an extremely high incidence of lung cancer. Abnormality of the fragile histidine triad (FHIT) gene has been proved to closely relate to lung cancer development. This study was to explore the loss of exons 3, 4, 5 and 8 of FHIT gene and FHIT protein expression in Yunnan Tin miners with lung cancer. METHODS: The exons 3, 4, 5, and 8 of FHIT gene in lung cancer cell line YTMCC of a Yunnan Tin miner, and 30 specimens of lung cancer from Yunnan Tin miners in Gejiu district and 22 specimens of lung cancer from non-miners in other regions were detected by polymerase chain reaction (PCR). The expression of FHIT protein in 90 specimens of human lung cancer and 43 specimens of lung cancer developed by the intratracheal instillation of Yunnan Tin mineral dust in F344 rats was detected by immunohistochemistry. RESULTS: Losses of exon 3 and exon 8 were detected in YTMCC cells. Lung cancer samples from Yunnan Tin miners and non-miners exhibited heterozygous loss of FHIT gene among exons 3, 4, 5 and 8. The percentages of FHIT gene deletion and loss of FHIT protein expression in Yunnan Tin miners with lung cancer were 68.2% and 70.6%, respectively. The percentages of FHIT gene heterozygous loss was significantly higher in lung cancer tissue than in normal lung tissue (P < 0.01). Loss of FHIT protein expression was detected in the early stage of F344 rat lung canceration after treatment of Yunnan tin Mineral dust:the percentage was 100% in 8 specimens of squamous dysplasia, carcinoma in situ, and early stage of squamous cell carcinoma. CONCLUSIONS: Deletion and abnormal expression of FHIT gene are common in Yunnan tin miners and non-miners with lung cancer. The high rate of loss of FHIT expression in precancerous lesions and lung cancer at early stage indicates that FHIT could be an early screening target of lung cancer.

[Establishment and biological characteristics of lung cancer cell line XWLC-05].

BACKGROUND & OBJECTIVE: The incidence of lung cancer is high at Xuanwei, Yunnan Province, at where the mortality rate of this disease in women is the highest in China. This study was to establish a Xuanwei woman lung adenocarcinoma cell line, and provide an in vitro experimental model for the study of preventing and treating lung cancer. METHODS: The cells derived from a surgical specimen of a woman patient with lung cancer were primarily cultured. The biological characteristics of the cell line were studied with light and electron microscopes, determination of doubling time and growth curve, culturing in soft agar, flow cytometry (FCM), chromosome and G-band detection, c-12 multiple tumor markers detection, and inoculation in mice. RESULTS: Morphologic study, proliferation dynamics, and invasive growth showed that the cultured cells have malignant characteristics. Their chromosome numbers ranged from 55 to 69, with a mode number of 60-63. The tumor formation rate in mice was 100% after axillary transplantation of the cells; the morphology of the tumor cells was similar to that of the pathologic specimen of the patient. The cell line was named XWLC-05. CONCLUSION: According to the newest rules of establishing a cell line in vitro, XWLC-05 is proved to be a new cell line of human lung adenocarcinoma.

[Comparison and analysis of expression of c-myc and p16 in cervical carcinoma].

BACKGROUND & OBJECTIVE: Activation of proto-oncogene and inactivation of tumor suppressor genes is related to the carcinogenesis of many tumors. It is still unclear whether abnormal expression of c-myc and p16 cooperate in the occurrence and progression of cervical carcinoma, and whether there exists a connection between the expression of two genes and the chemotherapy response of cervical carcinoma. This study was designed to investigate the correlation between the expression of c-myc and p16 and their roles in the genesis and development of the uterine cervical carcinoma and chemotherapy response. METHODS: Using in situ hybridization, 37 cases of cervical carcinoma (including 11 cases after chemotherapy), 21 cases of precancerous lesion and 5 cases of normal cervix were observed for c-myc and p16 mRNA with dig-labeled probes. An image analytic system was used to detect the gray degree values of the positive signals. RESULTS: The positive expression rates of p16 in normal cervix,CIN (cervical intraepithelial neoplasia) and cervical carcinoma were 100%, 71.4%, and 21.6%, respectively (P=0.0001), whereas the expression rates of c-myc were 0%, 42.9%, and 75.7% (P=0.0011), respectively. Statistically significant difference was found among the three groups for both p16 and c-myc. The expression of positive signals of c-myc increased with the increase of malignant degree, and the positive signals in CIN III were also higher than that in CIN II and CIN I. The expression rates of c-myc were decreased in cervical carcinoma after chemotherapy. There was a tendency of negative correlation between the expression of c-myc and p16(r(s)=-0.907). Expression of p16 and c-myc showed no significant difference between effectual and ineffectual chemotherapy groups. CONCLUSION: Both over expression of c-myc and descended expression of p16 may play an important role in the genesis and development of uterine cervical carcinoma. The increased expression of c-myc in different grade CIN suggests that carcinogenesis of cervix be progressive.